配合miScript PCR System定量检测成熟miRNA

  • 灵敏并特异性的定量检测miRNA
  • 对miRBase数据库中未列出的成熟miRNAs,提供用户订制服务
  • 可在GeneGlobe便利的订购
miScript Primer Assays是miRNA特异性引物,适合最新版本miRBase数据库中的所有人类、小鼠、大鼠和狗的miRNAs。miScript Primer Assays作为miScript PCR System的一部分,用于定量检测miRNA。cDNA使用miScript II RT Kit配合miScript HiSpec Buffer(只适用于成熟miRNA检测)或miScript HiFlex Buffer(使用相应的检测试剂同时定量检测各物种RNA)制备。cDNA作为模板,配合miScript Primer Assay和miScript SYBR Green PCR Kit进行real-time PCR。
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You can search for the following terms:
  • Entrez Gene IDs (e.g., 835)
  • RefSeq IDs (e.g., NM_032983, NP_116765)
  • Gene symbols (e.g., CASP2)
  • Cat. no. (e.g., SI00299551, QT01342509)
  • Sanger ID or Accession (e.g., hsa-let-7b, MI0000063)
  • CpG loci identification numbers (CG#) (e.g., CG17753661)

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When searching for miRNAs, do not omit the hyphens. Use hyphens or spaces (e.g., search for hsa-let-7b or hsa let 7b. Do not search for hsalet7b).

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Custom miScript Primer Assay (100)
10x Custom miScript Primer Assay (contains one miRNA-specific primer)
218200 Varies
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miScript Primer Assay (100)
10x miScript Primer Assay (contains one miRNA-specific primer)
218300 Varies
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miScript Primer Assays are intended for molecular biology applications. These products are not intended for the diagnosis, prevention, or treatment of a disease.


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Accurate detection of as low as 10 miRNA copies.|Broad dynamic range and high amplification efficiencies.|
Synthetic miR-21 was used to generate cDNA using the miScript II RT Kit with [A] miScript HiSpec Buffer or [B] miScript HiFlex Buffer (both provided in the kit). A range of amounts from 10 copies to 106 copies of this cDNA was used in real-time PCR using the miScript PCR System. Real-time PCR was performed on the Rotor-Gene Q cycler. The resultant CT values decreased linearly with increasing miRNA copy number, indicating sensitive detection from a wide range of template amounts.|Total RNA was purified from HeLa S3 cells using the miRNeasy Mini Kit. cDNA was prepared from a range of RNA amounts from 10 pg to 2 µg using the miScript II RT Kit with [A] miScript HiSpec Buffer or [B] miScript HiFlex Buffer (both provided in the kit). Each cDNA was used as a template in real-time PCR reactions using a miScript Primer Assays for miR-16, miR-20a, or miR-21. Linear standard curves demonstrated the broad dynamic detection range and high amplification efficiency of the miScript PCR System.|
Performance
miScript Primer Assays配合miScript PCR System检测成熟miRNA,结果可靠、重复性好。由于许多miRNAs表达水平低,灵敏度对数据的可靠性具有关键作用。miScript PCR System灵敏度高,可检测低至10拷贝的miRNA(参见"Accurate detection of as low as 10 miRNA copies")。只需10 pg的总RNA,即可得到准确、重复性好的miRNA数据(参见"Broad dynamic range and high amplification efficiencies")。持续高的扩增效率,确保使用∆∆CT方法进行多个miRNAs表达分析结果的准确性。
Principle

miScript PCR System配合SYBR® Green-based real-time PCR,灵敏和特异性定量检测和分析miRNA。miScript PCR System可以完成从RNA中合成cDNA和后续的real-time PCR检测miRNAs涉及的所有步骤。

使用miScript PCR System定量检测成熟miRNA,需要用到:

miScript II RT Kit,可简单、单步合成cDNA。单个cDNA合成反应可用于检测数以百计的miRNAs。双缓冲体系满足使用real-time PCR进行miRNA定量的独特要求。
miScript SYBR® Green PCR Kit,包含QuantiTect SYBR® Green PCR Master Mix和反向引物miScript Universal Primer,配合miScript Primer Assay或miScript miRNA PCR Array进行miRNAs检测。如需检测miRNA前体,无需使用miScript Universal Primer。
miScript Primer Assay,是miRNA特异性正向引物,配合miRBase数据库中的最新信息进行成熟miRNAs检测。 
Procedure

成熟miRNA检测实验中,使用miScript II RT Kit中的miScript HiSpec Buffer或miScript HiFlex Buffer,通过逆转录反应制备cDNA,作为real-time PCR模板。使用miRNA特异性的miScript Primer Assay(正向引物)和包含miScript Universal Primer(反向引物)和QuantiTect SYBR Green PCR Master Mix的miScript SYBR Green PCR Kit,进行real-time PCR反应。

其它非编码RNA检测实验中,使用miScript II RT Kit中的miScript HiFlex Buffer,通过逆转录反应制备cDNA,作为real-time PCR模板。使用miScript Primer Assay(正向引物)和包含miScript Universal Primer(反向引物)和QuantiTect SYBR Green PCR Master Mix的miScript SYBR Green PCR Kit,进行real-time PCR反应。如果没有针对目的小RNA的miScript Primer Assay,可以订制设计。

Applications

miScript Primer Assays作为miScript PCR System的一部分,用于:

  • 定量检测成熟miRNA
  • snoRNA检测

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Kit Handbooks
1
For real-time PCR analysis of microRNA using SYBR Green detection
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